Birth Defects Research (Part C) 90:103–109 (2010) REVIEW

Oxidative DNA Damage and Repair in Teratogenesis and Neurodevelopmental Deficits

Peter G. Wells,* Gordon P. McCallum, Kyla C. H. Lam, Jeffrey T. Henderson, and Stephanie L. Ondovcik

Several teratogenic agents, including ionizing radiation and xenobiotics area or the broader field of develop- such as phenytoin, benzo[a]pyrene, thalidomide, and methamphet- mental oxidative stress. The per- amine, can initiate the formation of reactive oxygen species (ROS) that spectives herein are largely illus- oxidatively damage cellular macromolecules including DNA. Oxidative trated by research using a limited DNA damage, and particularly the most prevalent 8-oxoguanine lesion, number of model teratogens from may adversely affect development, likely via alterations in gene tran- scription rather than via a mutational mechanism. Contributions from oxi- the authors’ laboratory, with apolo- dative DNA damage do not exclude roles for alternative mechanisms of gies to the many other investigators initiation like receptor-mediated processes or the formation of covalent workinginthisfield.Thisisan xenobiotic–macromolecular adducts, damage to other macromolecular emerging field with many questions targets like proteins and lipids, and other effects of ROS like altered signal remaining to be answered. transduction. Even in the absence of teratogen exposure, endogenous de- velopmental oxidative stress can have embryopathic consequences in the INTRODUCTION absence of key pathways for detoxifying ROS or repairing DNA damage. General Mechanisms of Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like Teratogenesis oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not For those teratogens that have always, protect the embryo from ROS-initiating teratogens. Protection been sufficiently studied, two gen- may be variably dependent upon such factors as the nature of the terato- eral types of mechanisms impli- gen and its concentration within the embryo, the stage of development, cated in abnormal development the species, strain, gender, target tissue and cell type, among other fac- involve receptor-mediated and tors. Birth Defects Research (Part C) 90:103–109, 2010. VC 2010 Wiley-Liss, Inc. reactive intermediate-mediated processes (Fig. 1) (Wells et al., Key words: oxidative DNA damage; DNA repair; reactive oxygen spe- 2009a). Although studies usually cies; teratogens; teratogenesis; neurodevelopmental deficits focus upon one hypothesis, these general mechanisms are not mutually exclusive nor are the PREAMBLE of oxidative DNA damage and repair various different pathways that in structural teratogenesis and neu- This commentary provides a brief constitute these general mecha- perspective on some of the ambigu- rodevelopmental deficits and is not nisms. For example, teratogens ities surrounding the potential role intended as a detailed review of this like the anticonvulsant drug phe-

Peter G. Wells is from the Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada and Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada Gordon P. McCallum is from the Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Kyla C. H. Lam is from the Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada Jeffrey T. Henderson and Stephanie L. Ondovcik are from the Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Abbreviations: 8-oxoG, 8-oxoguanine; ATM, ataxia telangiectasia mutated; BER, base excision repair; CSB, Cockayne syndrome B; CYPs, cytochromes P450; FPG, formamidopyrimidine glycosylase; G6PD, glucose-6-phosphate dehydrogenase; hOGG1, human oxo- guanine glycosylase 1; HR, homologous recombination; NER, nucleotide excision repair; OGG1, oxoguanine glycosylase 1; PHSs, prostaglandin H synthases; ROS, reactive oxygen species; SOD, superoxide dismutase Grant sponsor: National Institute of Environmental Health Science; Grant number: R21-ES013848; Grant sponsors: Canadian Insti- tutes of Health Research (CIHR), the National Cancer Institute of Canada, CIHR/Rx&D Health Research Foundation (Postdoctoral Fellowship), CIHR Frederick Banting and Charles Best Canada Graduate Scholarship *Correspondence to: Peter G. Wells, Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2. E-mail: [email protected] Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20177

VC 2010 Wiley-Liss, Inc. 104 WELLS ET AL.

Figure 1. Competing mechanisms potentially leading to teratogenesis. Changes in teratological outcomes resulting from modifying the pathways involved in DNA damage response and repair help distinguish the role of oxidative DNA damage from oxidative dam- age to other cellular macromolecules (protein, RNA, lipids, and carbohydrates) as well as from ROS effects via signal transduction. Similarly, changes in teratological outcomes due to modifications in antioxidants and antioxidative enzymes help distinguish the role of ROS from mechanisms involving electrophilic xenobiotic reactive intermediates and reversible, receptor-mediated interac- tions (from: Wells et al., 2009b).

nytoin and the environmental zo[a]pyrene may be bioactivated activation to a free radical inter- chemical benzo[a]pyrene may by enzymes like the cytochromes mediate, (2) redox cycling of a cause birth defects in part via P450 and prostaglandin H syn- quinone metabolite, (3) reperfu- both receptor- and reactive inter- thases to either an unstable elec- sion effects secondary to cardiac mediate-mediated mechanisms. trophilic or a free radical reactive suppression, (4) interference with Accordingly, results of a study intermediate, or to both (Wells the mitochondrial electron trans- implicating one mechanism often et al., 2009b). The former cova- port chain, and (5) induction or do not exclude the possibility of a lently (irreversibly) binds to cellu- activation of enzyme systems like lesser or greater contribution from lar macromolecules forming a NADPH oxidases (NOXs, DUOXs) additional mechanisms. The drug–macromolecular adduct, that produce superoxide and/or degree to which a given mecha- whereas the latter can initiate the hydrogen peroxide extracellularly nism or combination of mecha- formation of reactive oxygen spe- and/or within the cell in various nisms contribute to abnormal de- cies (ROS), including superoxide subcellular locales including the velopment may vary with such and hydrogen peroxide, which can nucleus (Fig. 2). As with the gen- factors as the nature of the terato- both alter signal transduction and eral mechanisms discussed above, gen, its concentration within the form the highly reactive hydroxyl these processes are not mutually embryo, the stage of develop- radical that can oxidatively dam- exclusive, and the contribution of ment, the species, strain, gender, age cellular macromolecules (Fig. one or more may vary with the target tissue and cell type, as well 2). The formation of drug–macro- same factors. At the macromolec- as environmental factors such as molecular adducts, oxidative mac- ular level, drug teratogens may diet, stress, and concomitant ex- romolecular damage, and ROS- form adducts with proteins and/or posure to xenobiotics. mediated altered signal transduc- DNA (or various RNAs), and drug- tion all may adversely affect em- initiated ROS may oxidatively bryonic and fetal development. damage proteins, DNA/RNA, and/ Reactive Intermediates Similarly, a given drug may initi- or lipids, the latter of which can In the case of reactive inter- ate the formation of ROS via sev- also result in lipid products that mediates, a given teratogenic eral mechanisms, including but react with proteins and/or DNA. xenobiotic like phenytoin or ben- not limited to: (1) enzymatic bio- The degree to which these various

Birth Defects Research (Part C) 90:103–109, (2010) OXIDATIVE DNA DAMAGE AND REPAIR 105

Figure 2. Biochemical pathways for the formation, detoxification, and cellular effects of xenobiotic free radical intermediates and reactive oxygen species (ROS). Abbreviations: Fe, iron; G-6-P, glucose-6-phosphate; GSH, glutathione; GSSG, glutathione disul- l 1 fide; H2O2, hydrogen peroxide; HO , hydroxyl radical; LPO, lipoxygenase; NADP , nicotinamide adenine dinucleotide phosphate; l O2 , superoxide; P450, cytochromes P450; PHS, prostaglandin H synthase, SOD, superoxide dismutase (modified from: Wells et al., 1997). types of chemical modifications constitute potential risk factors for T:A transversion mutations capa- and macromolecular targets con- genetic predisposition to endoge- ble of carcinogenic initiation (Fig. tribute to abnormal development nous causes of teratogenesis and 3) and hence may be involved in may vary for a given teratogen as in utero origins of adult diseases. the mechanism of transplacental it may for the two general types of Accordingly, the embryopathic carcinogenesis for some xenobiot- mechanisms discussed above. The potential of endogenous ROS in ics like diethylstilbestrol. However, same caveat holds for the multiple untreated animals is observed in this oxidative DNA lesion also has signal transduction cascades that genetically modified mice that are been causally implicated in struc- may be activated by ROS. deficient in a key antioxidative tural and functional teratogenesis, Although such teratological enzyme like glucose-6-phosphate as well as some neurodegenera- mechanisms are most frequently dehydrogenase (Nicol et al., 2000) tive processes, for which a muta- associated with xenobiotics like or a protein sensor like ataxia tional mechanism is less likely, but drugs and environmental chemi- telangiectasia mutated (ATM) that may involve alterations in gene cals, they can apply to other tera- detects DNA damage and transdu- transcription (Wells et al., 2009b). togenic agents like ionizing radia- ces the signal for DNA repair Agents discussed herein that may tion, which causes ROS formation (Bhuller and Wells, 2006). exert their teratogenic effects in and DNA damage including DNA part through the formation of 8- double-strand breaks. Also, the oxoG include phenytoin, benzo[a]- OXIDATIVE DNA DAMAGE contribution of ROS-initiated pyrene, thalidomide, metham- mechanisms elucidated in studies ROS and particularly hydroxyl rad- phetamine, and ionizing radiation, of chemical teratogenesis has icals can initiate the formation of although the latter is associated revealed ‘‘endogenous’’ mecha- more than 20 types of oxidative more with DNA double-strand nisms of abnormal development DNA damage, the most prevalent breaks. Although the 8-oxoG that may occur in the absence of of which is the 8-oxoguanine (8- lesion has been most commonly teratogen exposures, and hence oxoG) lesion that causes G:C to examined, it seems likely that

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levels of embryonic DNA damage lead to the transduction of a repair signal, whereas higher levels of damage lead to apoptosis. The major pathways involved in DNA repair include base excision repair of single-base damage, nu- cleotide excision repair of damage causing distortion to the DNA helix, and repair of DNA double- strand breaks by homologous recombination and nonhomolo- gous end joining (Fig. 5). This commentary will focus on the oxi- dative lesions, and particularly 8- oxoG, caused by the ROS-initiat- ing agents ionizing radiation, phenytoin, benzo[a]pyrene, and methamphetamine. The assign- ments to various pathways are tentative, because it is not known in many cases the full range of DNA lesions produced by a given Figure 3. Reaction of hydroxyl radicals (HOl) with guanine residues of DNA to form teratogen, the intrinsic teratogenic the molecular lesion 7,8-dihydro-8-oxoguanine (8-oxoguanine, 8-oxoG). If not (as distinct from mutational) effi- repaired, this oxidative damage can cause mutations and/or altered gene transcrip- cacy of such lesions, nor even tion, which may lead to cancer and/or embryopathies (from Wells et al., 2009b). which pathway(s) in the embryo may contribute to the repair of the lesions produced. For example, other oxidative DNA lesions, as dative stress could account for the there is in vitro evidence implicat- well as lesions formed via alterna- above association, and a causal ing homologous recombination as tive mechanisms, will prove to role for 8-oxoG in the mechanism a repair pathway protecting the have embryopathic consequences, of thalidomide teratogenesis cell from the cytotoxicity of phe- as is believed to be the case for remains to be established. nytoin, which induced both 8-oxoG ionizing radiation. The 8-oxoG formation and DNA double-strand lesion is implicated in embryo- breaks (Winn et al., 2003), and pathic mechanisms in part by its OXIDATIVE DNA DAMAGE for valproic acid, which caused elevation in embryonic tissues DETECTION AND REPAIR DNA double-strand breaks in the after agent exposure and by absence of 8-oxoG formation enhanced embryopathies in genet- Overview (Defoort et al., 2006). ically modified mice that lack key Although there is a great deal proteins involved in DNA damage known about DNA repair, we have DNA Damage Detection and detection and repair transduction, a limited understanding about the like p53 and ATM, or proteins explicit involvement of specific Repair in Teratogenesis directly involved in the repair of 8- repair pathways in protecting the The importance of DNA damage oxoG, like oxoguanine glycosylase embryo from DNA-damaging detection is supported by the 1 (OGG1) or Cockayne syndrome agents and particularly from ROS- enhanced teratogenicity of ben- B (CSB) protein (Wells et al., initiating agents. Two of the key zo[a]pyrene in p53 knockout mice 2009b). In the case of thalido- proteins known to be involved in (Nicol et al., 1995), and the mide, embryonic 8-oxoG forma- detection of DNA damage and potential embryopathic role for a tion is enhanced in the susceptible transduction of the signal for ROS-mediated oxidative lesion, as rabbit species, and not in the re- repair include p53 and ATM (Fig. distinct from a benzo[a]pyrene- sistant mouse species, consistent 4). However, interpretations of the DNA adduct, is suggested by the with a causal role for 8-oxoG in roles of these proteins may be ability of this xenobiotic to part because both that molecular confounded by the fact that, in increase embryonic 8-oxoG levels lesion and teratogenesis in rabbit addition to directing repair, they and the complete protection embryos are blocked by pretreat- also transduce the signal for cellu- against benzo[a]pyrene embryo- ment with the free radical spin lar apoptosis, and these two out- pathies in embryo culture by the trapping agent phenylbutylnitrone comes may have opposite conse- antioxidative enzyme superoxide (Parman et al., 1999). However, quences for embryonic develop- dismutase (Winn and Wells, other mechanisms related to oxi- ment. It is possible that lower 1997). p53 also protects against

Birth Defects Research (Part C) 90:103–109, (2010) OXIDATIVE DNA DAMAGE AND REPAIR 107

Figure 4. The potential relation of the ataxia telangiectasia mutated (ATM) and p53 proteins in the cellular DNA repair response or apoptosis after DNA damage initiated by oxidative stress (from Bhuller and Wells, 2006).

Figure 5. Major pathways for the repair of DNA damage caused by model teratogens. Abbreviations: APE1, apurinic/apyrimidinic endonuclease 1; B[a]P, benzo[a]pyrene; BER, base excision repair; CSA, Cockayne syndrome A protein; CSB, Cockayne syndrome B protein; DDB, damaged DNA-binding protein; DNA-PKCS, DNA protein kinase catalytic subunit; DNA pol, DNA polymerase; ERCC1, excision repair cross-complementing 1; FEN1, flap endonuclease 1; GGR, global genome repair; HR, homologous recombi- nation; HR23B, RAD23 homolog B; METH, methamphetamine; MMR, mismatch repair; MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; NER, nucleotide excision repair; NHEJ, nonhomologous end joining; OGG1, oxoguanine glycosy- lase 1; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RPA, replication protein A; TCR, transcription-coupled repair; TFIIH, transcription factor IIH; XPA-G, xeroderma pigmentosum A-G; XRCC1/4, X-ray cross-complementing 1/4 (modified from Wong, 2006). the teratogenicity of the activated for apoptosis (Wubah et al., Wells, 2006). The protective effect analog of cyclophosphamide 1996), illustrating the variable role of ATM against phenytoin terato- (Moallem and Hales, 1998), which of DNA damage detection path- genicity in vivo (Bhuller et al., initiates the formation of both ROS ways in teratogenesis. A similar 2006) was less comprehensive and drug–DNA adducts, so the protective role is observed with than that in embryo culture. precise mechanism of protection is ATM, evidenced by enhanced, With respect to DNA repair, the unclear. Conversely, p53 knockout gene dose-dependent teratogenic- most direct evidence of an embry- mice are protected against the ity in Atm knockout mice exposed opathic role for 8-oxoG comes ocular teratogenic effects of 2- to ionizing radiation in vivo from Ogg1 knockout mice, which chloro-20-deoxyadenosine, pre- (Laposa et al., 2004) or to phenyt- have deficient repair of the 8-oxoG sumably due to a reduced signal oin in embryo culture (Bhuller and lesion. In pregnant Ogg1 knockout

Birth Defects Research (Part C) 90:103–109, (2010) 108 WELLS ET AL. mice treated with the ROS-initiat- contribute to 8-oxoG repair via a oxidative stress in atm knockout mice. ing drug methamphetamine, the mechanism independent of OGG1, Toxicol Sci 93:146–155. Ogg1 knockout embryos exhibit as has been proposed by others Bhuller Y, Wells PG. 2006. A develop- mental role for ataxia-telangiectasia enhanced levels of 8-oxoG, and (Osterod et al., 2002). As with mutated in protecting the embryo the Ogg1 knockout offspring show Ogg1 mice, only female Csb from spontaneous and phenytoin- long-term postnatal neurodevelop- knockout mice showed signifi- enhanced embryopathies in culture. mental deficits compared with cantly enhanced susceptibility to Toxicol Sci 93:156–163. their heterozygous and wild-type methamphetamine-initiated neu- Defoort EN, Kim PM, Winn LM. 2006. Valproic acid increases conservative littermates (Wong et al., 2008). rodevelopmental deficits, although homologous recombination frequency The developmental importance of a similar nonsignificant trend was and reactive oxygen species forma- OGG1 is suggested by its twofold observed in male Csb knockout lit- tion: a potential mechanism for val- higher activity in fetal brain and termates. The CSB results are proic acid-induced neural tube defects. liver compared with maternal tis- consistent with the OGG1 results Mol Pharmacol 69:1304–1310. Jeng W, Wong AW, Ting-A-Kee R, sues (Wong et al., 2008). There in suggesting an embryopathic Wells PG. 2005. Methamphetamine- can be a remarkable gender de- role for the 8-oxoG lesion. enhanced embryonic oxidative DNA pendence in the developmental damage and neurodevelopmental deficits. Free Radic Biol Med 39:317– impact of ROS-initiated DNA EPILOGUE damage and repair. For example, 326. There is evidence that oxidative Laposa RR, Henderson JT, Xu E, Wells the enhanced susceptibility of PG. 2004. Atm-null mice exhibit methamphetamine-exposed Ogg1 DNA damage, and particularly the enhanced radiation-induced birth knockout mice was observed 8-oxoG lesion, may play a role in defects and a hybrid form of embry- only in female littermates (Wong the embryopathic mechanisms of onic programmed cell death indicat- ing a teratological suppressor func- et al., 2008), whereas a gender- endogenous oxidative stress and ROS formation enhanced by some tion for ATM. FASEB J 18:896–898. dependent susceptibility was not Moallem SA, Hales BF. 1998. The role observed in repair-normal outbred teratogenic agents. Although DNA of p53 and cell death by apoptosis CD-1 mice (Jeng et al., 2005). The repair can protect the embryo and necrosis in 4-hydroperoxycyclo- mechanism for this gender de- from such oxidative insults, this phosphamide-induced limb malfor- protection may vary with a variety mations. Development 125:3225– pendence has yet to be deter- 3234. mined. An embryopathic role for of factors, including the nature of Nicol CJ, Harrison ML, Laposa RR, et al. 8-oxoG is consistent with related the teratogen, its concentration 1995. A teratologic suppressor role studies in human embryonic kid- within the embryo, the stage of for p53 in benzo[a]pyrene-treated transgenic p53-deficient mice. Nat ney cells stably transfected with development, the species, strain, gender, target tissue and cell Genet 10:181–187. either human Ogg1 (hOgg1) or its Nicol CJ, Zielenski J, Tsui L-C, Wells bacterial homolog, formamidopyri- type, among other factors. Con- PG. 2000. An embryoprotective role midine glycosylase (Fpg) (Preston versely, in some cases, as when for glucose-6-phosphate dehydro- et al., 2007, 2009). Both hOgg1- dual pathways are transduced by genase in developmental oxidative proteins like p53 that regulate stress and chemical teratogenesis. and Fpg-transfected cells exhib- FASEB J 14:111–127. ited increased repair of the 8-oxoG pathways for repair and apoptosis, Osterod M, Larsen E, Le Page F, et al. lesion and resistance to the for- activation may increase the tera- 2002. A global DNA repair mecha- mation of 8-oxoG and cytotoxic togenicity of certain agents. In nism involving the Cockayne syn- drome B (CSB) gene product can effects caused by ROS (hydrogen other cases, teratogen-enhanced formation of intermediary products prevent the in vivo accumulation of peroxide) and ROS-initiating xeno- endogenous oxidative DNA base biotics including menadione, plati- in the repair pathway may prove damage. Oncogene 21:8232–8239. num drugs, and phenytoin. embryotoxic. Along with a more Parman T, Wiley MJ, Wells PG. 1999. A protective role for DNA repair comprehensive elucidation of the Free radical-mediated oxidative DNA activities and relevance of specific damage in the mechanism of thalido- also was evident in Csb knockout mide teratogenicity. Nat Med 5:582– mice, which were more susceptible repair pathways within the 585. than their heterozygous and wild- embryo, an expanded spectrum of Preston TJ, Henderson JT, McCallum type littermates to fetal 8-oxoG developmental conditions, terato- GP, Wells PG. 2007.Cellular models of altered base excision repair reveal formation and postnatal neurode- gens and structural and functional developmental outcomes will need a differential contribution of reactive velopmental deficits caused by oxygen species-induced 7,8-dihydro- methamphetamine, although less to be examined to provide a 8-oxo-20-deoxyguanosine to the cy- so than Ogg1 knockout mice (G.P. proper perspective of the role of totoxic mechanisms of platinum anti- McCallum, A.W. Wong, and P.G. oxidative DNA damage and repair cancer drugs cisplatin and oxalipla- in teratogenesis. tin. Paper presented at: Proceedings Wells, submitted). Although 8- of the FASEB Experimental Biology oxoG levels were higher in the Meeting, Washington, DC, Abstract methamphetamine-exposed Csb REFERENCES No. 765. knockout fetuses, CSB status did Preston TJ, Henderson JT, McCallum Bhuller Y, Jeng W, Wells PG. 2006. Vari- GP, Wells PG. 2009. Base excision not appear to alter the in vitro able in vivo embryoprotective role for repair of reactive oxygen species-ini- incision of 8-oxoG catalyzed by ataxia-telangiectasia-mutated against tiated 7,8-dihydro-8-oxo-20-deoxy- OGG1, suggesting that CSB may constitutive and phenytoin-enhanced guanosine inhibits the cytotoxicity of

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platinum anticancer drugs. Mol Can- togenesis, neurodevelopmental defi- Wong AW. 2006. The role of oxidative cer Ther 8:2015–2026. cits and cancer. Toxicol Sci 108:4– DNA damage and repair in metham- Wells PG, Kim PM, Laposa RR, et al. 18. phetamine-initiated neurodevelop- 1997. Oxidative damage and terato- Winn LM, Kim PM, Nickoloff JA. 2003. mental deficits. Toronto: University genesis. Mutat Res 396:65–78. Oxidative stress-induced homologous of Toronto. Wells PG, Lee CJJ, McCallum GP, et al. recombination as a novel mechanism Wong AW, McCallum GP, Jeng W, Wells 2009a. Receptor- and reactive inter- for phenytoin-initiated toxicity. J Phar- PG. 2008. Oxoguanine glycosylase 1 mediate-mediated mechanisms of macol Exp Ther 306:523–527. (OGG1) protects against metham- teratogenesis. In: Uetrecht JP, edito- Winn LM, Wells PG. 1997. Evidence phetamine-enhanced fetal brain oxi- r.Handbook of experimental pharma- for embryonic prostaglandin H syn- dative DNA damage and neurodeve- cology, Vol. 196: Mechanisms of thase-catalysed bioactivation and re- lopmental deficits. J Neurosci 28: adverse drug reactions. Heidelberg: active oxygen species-mediated oxi- 9047–9054. Springer. pp. 131–162. dation of cellular macromolecules in Wubah JA, Ibrahim MM, Gao X, et al. Wells PG, McCallum GP, Chen CS, phenytoin and benzo[a]pyrene tera- 1996. Teratogen-induced eye defects et al. 2009b. Oxidative stress in de- togenesis. Free Radic Biol Med 22: mediated by p53-dependent apopto- velopmental origins of disease: tera- 607–621. sis. Curr Biol 6:60–69.

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